Growth factor transduced cell-loaded ceramic scaffold for bone regeneration and repair

ABSTRACT

A method for repairing a bone defect of a patient includes providing a ceramic scaffold configured for filling the bone defect, loading the scaffold with growth factor transduced cells incorporating a gene that encodes a growth factor essential for bone formation, placing the ceramic scaffold with the growth factor transduced cells in or across the bone defect, and stabilizing the ceramic scaffold with the growth factor transduced cells in the patient until the bone defect is healed. An assembly for repairing a bone defect includes a ceramic scaffold configured for spanning the bone defect and a culture of live growth factor transduced cells incorporating a gene that encodes a growth factor essential for bone formation loaded onto the ceramic scaffold.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit and priority of U.S. provisionalpatent application Ser. No. 62/401,745 filed Sep. 29, 2016, which isincorporated herein in its entirety by reference.

GOVERNMENT LICENSE RIGHTS

This invention was made with U.S. government support under Contract#1335476 awarded by the National Science Foundation and under Contract#iR01AR057076-01A1 awarded by NIH. The U.S. government has certainrights in the invention.

FIELD

The present disclosure relates to methods and apparatus for repairingbone tissue, and more particularly to combined use of custom 3D-printedcalcium phosphate scaffolds and regional gene therapy in bone graftscenarios to heal critical sized bone defects.

BACKGROUND

Fracture non-union and inadequate bone formation in settings such astrauma, tumor, joint replacement and limb reconstructive surgeries areamong the most challenging problems in orthopedic surgery. Autologousbone graft is the gold standard to use in such situations, but itsdisadvantage is limited availability of the graft and complications andpain associated with graft harvest. Researchers have explored the optionof using precursor cells (from bone marrow, fat, muscle or othertissues) that have potential to transform into bone forming cells, butthe methods to purify these cells and potential of these cells to formbone are limited unless stimulated by the growth factors. Regional genetherapy is an attractive option as it potentially allows theinvestigator to incorporate the desired gene encoding the growth factoressential for bone formation into the host cells and implant these cellsback into the host at a particular site where they induce new boneformation.

Prior research has reported on the effect of regional gene therapy withbone morphogenetic protein-2-producing bone marrow cells on the repairof bone defects in rats, showing promise as one aspect of bonereplacement therapy. “3D printing” broadly understood as additivemanufacturing, has been proposed for forming scaffolds of calciumphosphate and collagen for bone regeneration, but not in conjunctionwith regional gene therapy. Additive manufacturing provides theadvantage of custom shaping for individual bone replacement therapy, butits suitability in conjunction with regional gene therapy is poorlyunderstood, if at all.

Bone regeneration in vivo or in vitro is desirable for providing morerapid and more effective clinical outcomes for treatment of severe boneinjury. It would be desirable, therefore, to provide more effectivemethods and apparatus for bone regeneration and replacement of lost bonetissue.

SUMMARY

This summary and the following detailed description should beinterpreted as complementary parts of an integrated disclosure, whichparts may include redundant subject matter and/or supplemental subjectmatter.

A method for repairing a bone defect of a patient may include providinga ceramic scaffold configured for spanning the bone defect. The methodmay further include loading the scaffold with one or more growth factortransduced cell lines (e.g., mesenchymal stem cells) incorporating agene that encodes a growth factor essential for bone formation. A cellline altered to incorporate a gene that encodes a growth factoressential for bone formation is referred to herein as a “growth factortransduced” cell line. The method may further include placing theceramic scaffold with the growth factor transduced cells in or acrossthe bone defect. The method may further include stabilizing the ceramicscaffold with the growth factor transduced cells in the patient untilthe bone defect is healed, using any suitable stabilizing technique.

In a related aspect, an assembly for repairing a bone defect may includea ceramic scaffold configured for spanning the bone defect and a cultureof live growth factor transduced cells incorporating a gene that encodesa growth factor essential for bone formation loaded onto the ceramicscaffold. The ceramic scaffold may be, or may include, a calciumphosphate material. In an aspect, the ceramic scaffold may include a 3Dprinted calcium phosphate material. The ceramic scaffold may shaped tomatch the bone in the areas adjacent to the defect, so as to fit closelyto the bone while spanning the defect.

To the accomplishment of the foregoing and related ends, one or moreexamples comprise the features hereinafter particularly pointed out inthe claims and fully described in the detailed description after thedrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing in vitro results of a trial using gene therapyin conjunction with a 3D printed ceramic scaffold.

FIG. 2 is a grayscale rendering of a photo illustrating cell viabilityof transduced cells on a 3D printed ceramic scaffold disk after 72hours.

FIG. 3 is an X-ray image showing results of a successful rat bone repairusing a method as disclosed herein.

FIG. 4 is a flowchart illustrating operations and aspects of a methodfor repair of a bone defect.

FIG. 5 is a perspective view showing an example of a 3D printed ceramicscaffold for bone repair.

FIG. 6 is a flow chart illustrating operations and aspects of making aceramic scaffold with the growth factor transduced cells.

DETAILED DESCRIPTION

The present disclosure concerns use of regional gene therapy withtransduced growth factor transduced cells in bone graft scenarios with3D-printed scaffolds. The combination of these technologies representsan innovative method of grafting bone with many potential clinicalapplications. The disclosure below describes experiments andexperimental results achieved by the inventors, followed by a summary ofthe novel subject matter underlying the experiments.

3D Printing

Calcium phosphate (CaP) scaffolds were 3D printed using a slurry-basedstereolithography process as developed by Dr. Song Chen et al. inCeramic Fabrication Using Mask-Image-Projection-based StereolithographyIntegrated with Tape-casting, Journal of Manufacturing Processes, 2015;20(3):456-464. Briefly, this 3D printing technique is performed by firstmixing a ceramic powder (CaP) with a photopolymer resin to create aslurry. A tape casting system is used to aid the recoating of eachslurry layer. A light source then activates the resin, curing it layerby layer until an object is built. The object, which is still a mixtureof ceramic and resin, is then heated in a furnace to burn out the resin.Since the resin has a much lower melting temperature than the ceramic,the ceramic part of interest is left behind as the final product.

In our experiments, Computer Aided Software (CAD) was first used tocreate a hollow elliptical cylinder 6 mm in length in order toapproximate the size and shape of a rat critical sized femoral defect.These scaffolds were 3D printed using commercially available calciumphosphate powder (Alfa Aesar #89836). An example of a resulting scaffold500 is shown in FIG. 5.

Regional Gene Therapy

Virk et al. 2011 (“Same day” ex-vivo regional gene therapy: a novelstrategy to enhance for bone repair. Mol Ther. 2011; 19:960-968)describes the gene therapy in detail. Briefly, a lentiviral based system(LV -BMP2) was created expressing bone morphogenetic protein 2 (BMP-2),Cultured rat bone marrow cells were transduced using a multiplicity ofinfection (MOI) of 25. These cells were used in the experiments detailedbelow.

Dr. J. R. Lieberman's and associates have focused on the development ofregional gene therapy to treat large-scale bone loss using a rodentmodel. His lab has demonstrated that a critical sized defect (the sizeat which the bone will not heal on its own) created in a rodent femurcan heal with the application of lentiviral transduced rat bone marrowcells (RBMC) by loading them on a commercially available carrier(collagen sponge or compression resistant matrix) and placing thecarrier in the defect (Virk et al. 2011).

Our experiments evaluated custom 3D-printed scaffolds combination withgenetically modified cells as disclosed in Virk et al, 2011.

Results

In Vitro BMP-2 Production

BMP-2 production of transduced RBMC was tested in vitro after 48 hoursand 14 days of cell culture on 15 mm diameter, 2 mm thick 3D printeddiscs. At 48 hours BMP-2 production was higher on 3D-printed scaffoldsas compared to control (Table 1).

TABLE 1 ng BMP-2 production per 10,000 cells 48 Hours 14 DaysNon-transduced RBMC 0.00 0.03 LV-BMP2 + 3D disc 1.71 48.62 LV-BMP2 1.26157.35

Based on our work, in vitro BMP-2 production on the 3D printed discs issufficient to heal a critically sized rat femoral defect.

In Vitro Cell Viability

Transduced rat bone marrow cells (RBMC) were cultured in vitro on top of3D printed CaP discs for 72 hours. Cell viability was determined using acommercially available Live/Dead assay kit (BioVision K501). Cellscultured on CaP disks demonstrated excellent viability at 72 hourscompared to a control (standard culture well). Cell viability on 3dprinted discs averaged 85% (SD 6%) relative to the control (FIG. 1 atgraph 100, FIG. 2 at photo 200). These cell viability results are higherthan a published study using comparable 3D printed calciumphosphate/collagen combination scaffolds (Inzana J, Olvera D, Fuller S,et al. 3D printing of composite calcium phosphate and collagen scaffoldsfor bone regeneration, Biomaterials. 2014; 35:4026-4034).

In Vivo Bone Formation

A pilot experiment using a 12-week-old Lewis rat was performed. Astandard 6-mm mid-diaphyseal femoral defect was created as described inprior publications (Alae, F., Liebermen, J. R., et al., Biodistributionof LV-TSTA transduced rat bone marrow cells used for “ex-vivo” regionalgene therapy for bone repair. Curr Gene Ther. 2015; 15(5):481-491, andVirk et al. 2011). A 3D printed CaP scaffold loaded with 5 millionlentiviral transduced rat bone marrow cells was placed in the defect.The defect was healed 8 weeks after the surgical procedure (FIG. 3 atx-ray image 300). The 3D printed CaP scaffold 310 itself and theregenerated “bridging bone” 320 are clearly visible and pointed out inFIG. 3, A drawing of a model for a similar 3D printed CaP scaffold 500is shown in FIG. 5. FIG. 2 shows a culture 200 of mesenchymal stem cellsincorporating a gene that encodes a growth factor essential for boneformation, grown on a CaP ceramic disk.

Scaffold Shaping and Configuration; Results

More recently, Computed Tomography (CT) data from an intact rodent femurwas obtained from our labs prior work. Commercially available software(Mimics; Materialise NV, Leuven, Belgium) was then used to convert a 6mm section of diaphyseal bone into a file type compatible with 3Dprinting software, resulting in a model of a scaffold 500 as shown inFIG. 5. Additionally, 700-micrometer holes 510 were added to the modelscaffold to facilitate cellular growth and communication.

An alternative ceramic powder, beta Tri-Calcium Phosphate (beta TOP),may also be used to 3D print the scaffolds. We have 3D printed scaffoldsbased on the “rodent specific” CT data, These may be tested followingsimilar methods as described above.

Prior to surgical implantation, the scaffold and loaded cells form anassembly made of a ceramic scaffold 500 configured for spanning the bonedefect, and a culture of live mesenchymal stem cells 200 or other growthfactor transduced cells incorporating a gene that encodes a growthfactor essential for bone formation loaded onto the ceramic scaffold.Suitable cells for being transduced with one or more genes that encode agrowth factor may include, for example, mesenchymal cells, bone marrowcells, fibroblasts, adipose-derived cells, umbilical cord cells, ormuscle cells. The ceramic material may include, for example, CaP or betaTCP. In a clinical setting, the assembly may be prepared in advance ofsurgery and maintained alive in vitro until surgical implantation. In analternative, the surgeon may load live growth factor transduced cells onthe ceramic scaffold for the first time after it is in place in thepatient's body (in vivo), or may supplement an in vitro loading of cellswith a second application in vivo.

The scaffold may be thinner than the bone wall to be repaired andperforated with circular openings 510 in the range of about 300 to 1000microns, for example, about 500 to 700 microns, or about 700 microns.The openings 510 may be spaced as desired to facilitate bone regrowth,for example, uniformly or semi-uniformly center-to-center spaced atabout 1.5 to 5 times the opening's largest diameter. The scaffold isgenerally tube-shaped with an interior surface 520 and exterior surface530. The assembly of scaffold and live growth factor transduced cellsenables regeneration of structural bone tissue from the loaded cellculture on its openings 510, exterior 530, interior 520 and byrecruitment of local progenitor cells.

Flowcharts and Methods

Referring to FIG. 4, a method 400 for repairing a bone defect of apatient may include, at 410, providing a ceramic scaffold configured forspanning the bone defect, for example, by 3D printing a CaP or otherceramic scaffold or by obtaining a pre-printed scaffold from amanufacturer sized to span the bone defect, for example based on apreceding CT scan as for the rodent femur described above. The method400 may further include, at 420, loading the scaffold with growth factortransduced cells incorporating a gene that encodes a growth factoressential for bone formation, for example, by preparing or obtaining aculture of lentiviral transduced bone marrow cells as described herein,applying the culture to the scaffold, and confirming viability of theculture loaded on the scaffold prior to implantation. For example, thescaffold with pre-loaded mesenchymal cells as described may be obtainedin the form of a prepared assembly from an independent source, e.g., aspecialized laboratory, or the cell preparation and loading of thescaffold may be performed by a laboratory controlled by the facilityperforming the scaffold-implantation surgery. The method may furtherinclude, at 430, placing the ceramic scaffold with the growth factortransduced cells in or across the bone defect.

The method 400 may further include, at 440, stabilizing the ceramicscaffold with the growth factor transduced cells in the patient untilthe bone defect is healed, using any suitable stabilizing technique. Theceramic scaffold is semi-structural and designed for load sharing. Thescaffold may not be strong enough by itself to stabilize the defectwithout load sharing from other structural members. Depending on thenature of the defect, it may be stabilized using bio-compatible metalplates, rods, or other suitable structural members.

The enumerated operations 410, 420, 430, 440 may be performed in anyoperable order with suitable modifications. For example, the operation430 placing the ceramic scaffold in or across the defect may beperformed, but without first loading growth factor transduced cells ontothe ceramic. Then, the operation 420 of loading the scaffold with growthfactor transduced cells may be performed while the scaffold is in placearound the defect. For example, the growth factor transduced cells maybe suspended in a bio-compatible fluid and applied to the ceramicscaffold in vivo or in vitro, using a pipette or other suitable fluidapplicator,

Referring to FIGS. 5 and 6, further aspects of the present disclosuremay include a method 600 for making a ceramic scaffold 500 loaded withgrowth factor transduced cells as described herein, for use in themethod 400 or other suitable method. The method 600 may include, at 610,characterizing a specific bone defect of a patient. For example, theoperation 610 may include scanning a bone defect by a 3D scanner, forexample, a CT scanner, digitizing 3D information obtained by thescanning, and associating the digitized information with an identifierfor the patient and defect site. In an alternative, the characterizing610 may include receiving information that associates an identifier fora patient and/or bone defect with 3D information relating to the defector to a scaffold for bridging the defect.

Further aspects of the method 600 may include, at 620, providing a 3Dmodel of a scaffold for bridging the bone defect characterized by thefirst operation 610. As used herein, “providing” includes but is notlimited to engaging a person or entity to create the 3D model based onthe data characterizing the bone defect. For example, a medicaltechnician may design a 3D model to fit stable portions of the bone thatare expected to remain as scanned after the bone defect is prepared forrepair, e.g., by cleaning out damaged tissue. The stable regions of thebone may be adjacent to defective regions of the bone.

The method 600 may further include, at 630, providing a ceramic scaffoldthat includes calcium and phosphate or that consists essentially ofcalcium and phosphate materials, based on the 3D model provided by thepreceding operation 620. Providing may include manufacturing the ceramicscaffold, or obtaining the ceramic scaffold from a manufacturer orsupplier. A slurry-based stereolithography 3D printing method asdescribed by Dr. Song Chen (see reference herein above) may be used tomanufacture the ceramic scaffold. The manufacturing may include mixing apowdered calcium-phosphate ceramic or pre-ceramic material with aphotopolymer resin to create a slurry as feedstock for astereolithographic additive manufacturing process (e.g., using tapecasting as described above), thereby forming a “green” prefiredscaffold. The green scaffold, which may include a mixture of ceramic andorganic resin and/or a pre-ceramic polymer, may be heated under suitableconditions (e.g., oxidizing or non-oxidizing, depending on the processused) to purge non-ceramic materials and/or transform a pre-ceramicmaterial into a ceramic material, at 630, forming the ceramic scaffold500 made of a calcium phosphate material, alone or in combination withother materials. Likewise, the method may include manufacturing thescaffold using a 3D printing technique as described herein above, orother suitable method. 3D printing may be especially advantageous forforming the ceramic-resin scaffold when it is desired to custom shapethe ceramic-resin scaffold to match an individual morphology of the bonein healthy areas surrounding the defect. For example, the ceramic-resinscaffold may be formed to have an inner surface 520 matching an outersurface of the healthy bone areas. Forming the ceramic-resin scaffold500 may also include forming a plurality of holes 510 having diametersin the range of about 300 μ to 1000 μ (e.g., in the range of 500 μ to700 μ, or about 700 μ) in the ceramic-resin scaffold, for example duringthe 3D printing process.

The method 600 may further include, at 640, loading the ceramic scaffoldwith live growth factor transduced cells incorporating a gene thatencodes a growth factor essential for bone formation. As discussedabove, loading may be performed in vitro, in vivo, or both. In anotheraspect, the growth factor transduced cells may be prepared forincorporating the gene that encodes the growth factor by transducing thegene into the growth factor transduced cells. For example, the methodmay include preparing the growth factor transduced cells incorporatingthe gene that encodes the growth factor by a lentiviral basedtranscriptional activation system expressing bone morphogenetic protein2.

Having thus described embodiments of methods and apparatus for repairinga bone defect or providing a ceramic scaffold loaded with growth factortransduced cells, it should also be appreciated that variousmodifications, adaptations, and alternative embodiments thereof may bemade within the scope and spirit of the present invention. For example,3D printed calcium ceramic scaffolds have been disclosed, but theinventive concepts described above may be equally applicable toscaffolds of other ceramic materials, or scaffolds made by othermanufacturing methods than disclosed herein above. In addition, aculture of growth factor transduced cells incorporating a gene thatencodes a growth factor essential for bone formation may be prepared byany suitable method whether or not described herein.

1. A method for repairing a bone defect of a patient, comprising:providing a ceramic scaffold configured for filling the bone defect;loading the scaffold with growth factor transduced cells incorporating agene that encodes a growth factor essential for bone formation; placingthe ceramic scaffold with the growth factor transduced cells in oracross the bone defect; and stabilizing the ceramic scaffold with thegrowth factor transduced cells in the patient until the bone defect ishealed.
 2. The method of claim 1, further comprising forming the ceramicscaffold by 3D printing a calcium phosphate material.
 3. The method ofclaim 3, wherein forming the ceramic scaffold further comprisespreparing a slurry including a calcium phosphate powder and a resin,forming a ceramic-resin scaffold by 3D-printing the slurry, and removingthe resin from the ceramic-resin scaffold by heating.
 4. The method ofclaim 4, wherein forming the ceramic-resin scaffold comprises shapingthe ceramic-resin scaffold to match undamaged areas adjacent to thedefect, so as to fit against the undamaged areas while spanning thedefect.
 5. The method of claim 1, further comprising preparing thegrowth factor transduced cells incorporating the gene that encodes thegrowth factor by transducing the gene into the growth factor transducedcells.
 6. An assembly for repairing a bone defect, comprising: a ceramicscaffold configured for spanning the bone defect; and a culture of livegrowth factor transduced cells incorporating a gene that encodes agrowth factor essential for bone formation loaded onto the ceramicscaffold.
 7. The assembly of claim 6, wherein the ceramic scaffoldcomprises a calcium phosphate material.
 8. The assembly of claim 6,wherein the ceramic scaffold comprises a 3D printed calcium phosphatematerial.
 9. The assembly of claim 6, wherein the ceramic scaffold isshaped to match the bone in undamaged areas adjacent to the defect, soas to fit against the undamaged areas while spanning the defect.
 10. Theassembly of claim 6 wherein the ceramic scaffold comprises a pluralityof holes in the range of 300 μ to 1000 μ there through.
 11. The assemblyof claim 6, wherein the growth factor transduced cells incorporating thegene that encodes the growth factor are prepared by transducing the geneinto the growth factor transduced cells.
 12. The assembly of claim 6,wherein the growth factor transduced cells are selected from mesenchymalcells or bone marrow cells.
 13. A method for repairing a bone defect ofa patient, comprising: characterizing a bone defect; providing a 3Dmodel of a scaffold for bridging the bone defect; providing a ceramicscaffold comprising calcium and phosphate based on the 3D model; andloading the ceramic scaffold with live growth factor transduced cellsincorporating a gene that encodes a growth factor essential for boneformation.
 14. The method of claim 13, further comprising forming theceramic scaffold by 3D printing a calcium phosphate material.
 15. Themethod of claim 14, wherein forming the ceramic scaffold furthercomprises preparing a slurry including a calcium phosphate powder and aresin, forming a ceramic-resin scaffold by 3D-printing the slurry, andremoving the resin from the ceramic-resin scaffold by heating.
 16. Themethod of claim 13, wherein providing the 3D model comprises shaping 3Dmodel for causing the ceramic scaffold to match undamaged areas adjacentto the bone defect, so as to fit against the undamaged areas whilespanning the bone defect.
 17. The method of claim 13, wherein providingthe ceramic scaffold comprises forming a plurality of holes in the rangeof 300 μ to 1000 μ in the ceramic scaffold.
 18. The method of claim 13,further comprising preparing the growth factor transduced cellsincorporating the gene that encodes the growth factor by transducing thegene into the growth factor transduced cells.
 19. The method of claim13, further comprising preparing the growth factor transduced cellsincorporating the gene that encodes the growth factor by a lentiviralbased transcriptional activation system expressing bone morphogeneticprotein
 2. 20. The method of claim 13, wherein the growth factortransduced cells are selected from mesenchymal cells or bone marrowcells.